This CounterAct R21 project focuses on early-stage development of a novel nanoparticle-based drug delivery system for sustained and extended release of the FDA-approved naloxone as a strategy to reduce fatal overdoses from fentanyl and its analogs. Proposed activities will include optimization of the naloxone-nanoparticle (NLX-NP) formulation and testing its efficacy in pre-clinical models of exposure to fentanyl. The specter of a fentanyl-based Mass Casualty Incident has been raised in recent years due to the increase incidence of accidental poisoning due to fentanyl or its analogs. Fentanyl is a potent hydrophobic small molecule that can cause poisoning upon ingestion of less than 2-3 mg of this compound. The current countermeasure for a fentanyl poisoning is the administration of multiple doses of a mu opioid receptor antagonist such as naloxone. Unfortunately, the circulatory half-life of fentanyl is greater than that of the antidote due to fentanyl?s absorption into adipose tissue, which act as a drug eluting reservoir increasing the likelihood of re-narcotization. Hence, fentanyl can still act as a poison long after the naloxone has been metabolized and excreted. To more effectively reverse the lethality of fentanyl and its derivatives, new antidotes or antidote delivery strategies are required. This R21 project tests the hypothesis that the circulatory half-life of an antagonist can be increased through the use of a novel drug delivery system consisting of naloxone covalently bound and incorporated into biodegradable nanoparticles (NLX-NP). The NLX-NP system allows for the linear sustained release of therapeutic doses of the FDA-approved naloxone. AIM1 will test how the composition of the nanoparticle-based delivery platform impacts the sustained release of naloxone in rats. AIM2 will test whether the NLX-NP will reverse or reduce fentanyl-induced pharmacological effects, including respiratory depression in rats. The NLX-NP will be delivered intramuscularly, which is the route of administration that best reflects field conditions for delivery of countermeasures to opioids or other chemical threats.